The present invention relates generally to the field of natural product extraction. More particularly, it concerns the use of supercritical fluid for the extraction of carotenoids from green plant materials.
Carotenoids are highly colored naturally occurring compounds, which are widely distributed in nature. Carotenoids may be classified as hydrocarbon carotenes or xanthophylls, which are oxygenated derivatives of carotenes. Representative examples of carotenes include β-carotene, alpha-carotene, and lycopene. Examples of xanthophylls include lutein, astaxanthin, canthaxanthin, zeaxanthin, and capsorubin. Carotenoids have been shown to have anti-oxidant properties and have been studied for the prevention of cancer and other human diseases.
Carotenoids are naturally present in edible leaves, flowers, and fruits, and are readily obtained from flowers (i.e. marigold), berries, and root tissue (i.e. carrots). Hydrocarbon carotenes, such as β-carotene and lycopene, are typically present in an uncombined free form, which is entrapped within chloroplast bodies within plant cells. Xanthophylls, such as lutein, are abundant in a number of yellow or orange fruits and vegetables such as peaches, mango, papaya, prunes, acorn squash, and oranges. Some Xanthophylls are present in plant flowers, such as marigolds, as long chain fatty esters, typically diesters, of acids such as palmitic and myristic acids. Generally, the free forms of carotenoids are present in the chlorplasts of green plants such as alfalfa, spinach, kale and leafy green plant materials. The free form of the carotinoids provides better adsorption when consumed in foods or as a supplement.
Lutein is a xanthophyll found in high concentrations in the macula of the eye and in the central part of the retina. It serves important roles in vision to help filter ultraviolet wavelengths of light to prevent damage to the eye lens and macula. Lutein's antioxidant properties are believed to help protect the macula, which is rich in polyunsaturated fats, from light-induced free radicals. Lutein can not be produced by the body, and consequently, must be ingested. Thus, lutein has become increasingly used in nutritional supplements for the prevention and/or treatment of vision losses due to macular degeneration, cataracts and retinitis pigmentosa.
Lutein has been shown to have significant potential in the prevention of age-related macular degeneration (AMD), the leading cause of irreversible blindness among Americans age 65 and older. Lutein helps build macular pigment density, a critical factor in the health of the macula and the retina. It has been found that high intake of lutein-rich green plants (spinach and kale) reduced the rate of AMD by 40% whereas Beta-carotene, vitamin A, zinc, and vitamin E were not seen to have an effect (Seddon et al. 1994). It has been shown that the accumulation of lutein in the macular pigment is dependent upon dietary intake and that the density of the macular pigment is related to the preservation of visual sensitivity and protection from AMD (Pratt, 1999, Richer, 2001). Other vision loss problems, such as cataracts and retinitis pigmentosa may also be stopped or reduced with a high intake of lutein.
The most common source of extracted lutein is from marigold flower petals, which contain one of the highest levels of lutein known and have a low concentration of other carotenoids. Methods of the purification of lutein-fatty acid esters from marigold flower petals are reported in U.S. Pat. Nos. 4,048,203, 5,382,714 and 5,648,564, in which dried ground marigold flower petals are extracted with a hydrocarbon solvent. In U.S. Pat. No. 5,648,564, extraction is performed 8-10 times with a 60-minute soak in hexane solvent for the extraction of the carotenoid from the marigold, and uses 320-400 L hexane for each 1 kg of dried marigold flower petals. The solvent is removed and the residue is dissolved in a hot alcohol. The solution is then filtered and then the lutein fatty acid ester is precipitated out. To obtain a more digestible form of lutein from extracted marigold flower petals, the extract is saponified at high pH (10+) or hydrolyzed to convert the product to a free form lutein.
U.S. Pat. No. 5,382,714, reports using commercially available saponified marigold oleoresins to crystallize lutein after saponification of the oleoresins using organic solvents. Purification of lutein from saponified marigold oleoresins without the use of added organic solvents is reported in U.S. Pat. No. 5,648,564.
There are several drawbacks to the extraction methods reported above. For example, the method reported in U.S. Pat. No. 5,648,564 uses caustic, high pH conditions that may be dangerous and may cause yield losses and vapor exposure, as well as producing toxic waste materials that need to be disposed of when completed. Trace amounts of these toxic chemicals and solvents may be present in the final products, which may be a problem for use of the resulting lutein extract for human consumption. The method reported in U.S. Pat. No. 5,382,714 uses organic and caustic solvents such as hexane, propane diol, and potassium hydroxide for extraction and saponification processes, which may not be totally removed during the purification process. Furthermore, neither method utilizes a starting material in which lutein is obtained in its free form. As previously noted, free form carotenoids such as lutein may provide better adsorption into the body during consumption. Thus, it would be desirable to provide a lutein extraction method that isolates the free form lutein without requiring the use of organic solvents during any steps, from the extraction of lutein from raw materials to the production of free lutein for consumption.
Lutein is abundantly present in a free, non-esterified form in green plants such as alfalfa, broccoli, green beans, green peas, lima beans, cabbage, kale, spinach, collards, mustard greens, turnip greens, kiwi, and honeydew. Green plants may also be rich in a variety of additional nutrients. For example, alfalfa is rich in proteins, minerals, and vitamins. It contains all 21 amino acids, and has significant concentrations of vitamins A, D, E, B-6, and K, calcium, magnesium, chlorophyll, linolenic and linoleic fatty acids, phytoestrogens, phosphorous, iron, potassium, trace minerals and several digestive enzymes. It also contains several saponins, many sterols, flavonoids, coumarins, alkaloids, acids, additional vitamins, amino acids, natural sugars, proteins (25% by weight), minerals, trace elements and other essential nutrients.
Extraction of lutein from green plants may be beneficial because it removes the need for the additional chemical step of saponification or ester cleavage to release the free lutein, which is the desired form for best absorption as consumed. However, the isolation and purification of lutein from plants has not been economical in the past because many expensive and time-consuming purification steps have been required to separate the lutein from the large quantities of other compounds present in the plant materials.
Supercritical fluids (SCF), which are gases above their critical pressure and temperature, have been used in certain industries to perform extractions. SCFs are dense gasses in a separate phase, which is distinct from normal gas phase. SCFs have a density and solvating power similar to that of a liquid and diffusion rates similar to that of a gas. Supercritical fluids are unlike liquids because their solvent power is highly sensitive to pressure changes and may be varied over wide limits by changing the pressure.
SCF extraction offers a relatively rapid, simple and inexpensive technique to perform purification or compound preparations. Most compounds, once dissolved, can quickly and cleanly be precipitated or removed from the supercritical fluids by lowering the pressure and/or temperature or both to achieve separation. Because a slight change in the pressure or temperature of a system causes significant change in solubility, the use of SCF enables a highly efficient isolation procedure of the desired components to be extracted. Using the method of post-extraction fractionation with a column designed to allow for temperature and pressure drops at different levels to gain the desired results may effect further concentration and purification.
One method of extracting carotenoids such as lutein from alfalfa without using toxic solvents is reported in Favati et al., Supercritical CO2 Extraction of Carotene and Lutein from Leaf Protein Concentrates (1988). In the method reported in Favati, extracts containing mixtures of free lutein and β-carotene were obtained from alfalfa by supercritical extraction in a single stage extractor. This laboratory scale extraction was done in a single step, extracting a mixture of lutein, carotene, and other components from a leaf protein concentrate, with the relative concentrations of the two carotenoids dependent upon the extraction pressure used. The carotenoid content obtained from the process was 1.5% of the total extract.
Although the supercritical extraction method reported in Favati et al. overcomes the aforementioned problems with the health and safety risks of conventional solvent extractions, the resulting extracts include an uncontrolled mixture of lutein and other carotenoids in a single extraction. However, given the beneficial health effects of lutein, it would be desirable to obtain an isolated lutein extract containing a substantial concentration of lutein while being substantially free of other carotenoids. It may also be beneficial to obtain extracts with controlled concentrations of lutein and other desired nutrients such as β-carotene and/or fatty acids in order to treat patients with varying nutritional needs based on age (e.g., adults versus children) and/or the existence of eye conditions such as macular degeneration.